Methods for using enriched exosomes as a platform for monitoring organ status

Abstract

This present disclosure relates to the use of one or more biomarkers to monitor the conditional state of an organ or tissue, including transplanted tissue, or disease state, including diabetes, in a biological sample of a subject. Accordingly, this disclosure provides for: methods and kits for determining the presence of one or more biomarkers for organ or tissue rejection/injury or diabetes in a biological sample of a subject; methods for using the presence of such biomarkers to predict or diagnose organ or tissue rejection/injury or diabetes in a subject; and methods to select or modify a therapeutic regimen for a subject based on the use of such biomarkers.

What is claimed is: 
     
         1 . A method for monitoring transplanted organ or tissue rejection and/or injury in a subject, comprising:
 (a) obtaining a biological sample from a subject;   (b) at least one of isolating, purifying, or identifying a donor organ- or tissue-derived microvesicle from the biological sample; and   (c) detecting a level of at least one biomarker associated with the donor organ-or tissue-derived microvesicles;   wherein the change in the presence and/or level of the at least one biomarkers allows for monitoring of transplant organ or tissue rejection and/or injury of the subject.   
     
     
         2 . The method of  claim 1 , further comprising detecting a level of at least one biomarker in a first biological sample obtained from the subject prior to a therapy; and detecting a level of at least one biomarker in a second biological sample obtained from the subject, at one or more points during therapy for assessing the efficacy of the therapy,
 wherein the therapy is efficacious for preventing or treating organ or tissue rejection and/or injury in the subject when there is a change in the level of the at least one biomarker in the second or subsequent samples, relative to the first sample.   
     
     
         3 . The method of  claim 1 , wherein the biomarker is selected from the group consisting of a protein, a nucleic acid, a pool of one or more donor organ- or tissue-derived microvesicles, and a pool of one or more recipient derived microvesicles. 
     
     
         4 . The method of  claim 3 , wherein the protein is heat shock cognate protein (Hsc-70), angiopoietin-1, hemopexin, complement C3, ZnT8, GAD65, or combinations thereof. 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 3 , wherein the pool of one or more donor organ- or tissue-derived microvesicles is FXYD2 or major histocompatibility complex protein expressing microvesicles. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 3 , wherein the pool of one or more recipient derived microvesicles is a cluster of differentiation protein expressing microvesicles. 
     
     
         10 . The method of  claim 3 , wherein a change in the size and/or number of the donor organ- or tissue-derived microvesicles in the pool of donor organ- or tissue-derived microvesicles indicates transplant organ or tissue rejection and/or injury. 
     
     
         11 . The method of  claim 10 , wherein the pool is insulin expressing microvesicles. 
     
     
         12 . The method of  claim 10 , wherein a decrease in the number of the donor organ- or tissue-derived microvesicles in the pool of donor organ- or tissue-derived microvesicles indicates transplant organ or tissue rejection and/or injury. 
     
     
         13 . The method of  claim 12 , wherein an about 0.20 to about 5 times decrease in the number of organ- or tissue-derived microvesicles as compared to a subject not undergoing rejection indicates that the subject is beginning to reject the organ. 
     
     
         14 . The method of  claim 13 , wherein the decrease is about 0.3 times. 
     
     
         15 . The method of  claim 1 , wherein the subject is human. 
     
     
         16 . The method of  claim 1 , wherein the biological sample is a blood or urine sample. 
     
     
         17 . The method of  claim 1 , wherein the donor organ or tissue is pancreatic islet or kidney. 
     
     
         18 . The method of  claim 1 , wherein the method comprises contacting the biological sample with an antibody specific for a protein to isolate, purify, or identify the donor organ- or tissue-derived microvesicle from the biological sample. 
     
     
         19 . The method of  claim 18 , wherein the protein is a surface protein. 
     
     
         20 . The method of  claim 19 , wherein the surface protein is selected from the group consisting of a major histocompatibility complex protein, FXYD2, and a cluster of differentiation protein. 
     
     
         21 . The method of  claim 20 , wherein the major histocompatibility complex protein is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR or a combination thereof. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 18 , wherein the antibody is conjugated with magnetic beads. 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 24 , wherein the biological sample is further enriched comprising contacting the biological sample with magnetic beads conjugated with an antibody specific for the surface protein FXYD2. 
     
     
         32 . A method for diagnosing diabetes and/or predicting a risk of diabetes in a subject, comprising
 (a) obtaining a biological sample from a subject;   (b) at least one of isolating, purifying, or identifying a beta islet derived microvesicle from the biological sample;   (c) detecting one or more biomarkers from the biological sample associated with the beta islet derived microvesicles; and   (d) diagnosing diabetes in the subject,   wherein a change in the presence and/or level of the one or more biomarkers as compared to a healthy subject indicates that the subject has or is developing diabetes.   
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 1 , further comprising determining a level of one or more biomarker in a first biological sample obtained from the subject prior to a therapy; and determining the presence or level of the one or more biomarker in a second biological sample obtained from the subject, at one or more points during the therapy, wherein a change in the level of the one or more biomarkers in the second or subsequent samples, relative to the first sample indicates that there is a change in a diabetic status of the subi ect. 
     
     
         35 . The method of  claim 34 , wherein the therapy is efficacious for treating diabetes in the subject when there is a change in the level of the one or more biomarkers in a second or subsequent samples, relative to a first sample. 
     
     
         36 . The method of  claim 32 , wherein the biomarker is selected from the group consisting of a protein, a nucleic acid, a pool of one or more subject derived microvesicles. 
     
     
         37 . The method of  claim 36 , wherein the protein is selected from the group consisting of FXYD2, heat shock cognate protein 71 (Hsc-70), angiopoietin-1, hemopexin, complement C3, ZnT8, GAD65, and combinations thereof. 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . The method of  claim 36 , wherein a change in the size and/or number of the donor organ- or tissue-derived microvesicles in the pool of subject derived microvesicles indicates that the subject has or is developing diabetes. 
     
     
         42 . The method of  claim 32 , wherein the subject is human. 
     
     
         43 . The method of  claim 32 , wherein the 
     
     
         44 . The method of  claim 32 , wherein the method comprises contacting the biological sample with magnetic beads conjugated with an antibody specific for a biomarker to isolate, purify, or identify the subject derived microvesicle from the biological sample. 
     
     
         45 . The method of  claim 32 , wherein the subject derived microvesicles are beta islet exosomes. 
     
     
         46 . A method for enriching organ- or tissue-derived microvesicles, comprising:
 (a) obtaining a biological sample from the subject; and   (b) contacting the biological sample with an antibody specific for a protein to isolate, purify, or identify the donor organ- or tissue-derived microvesicle from the biological sample.   
     
     
         47 . The method of  claim 46 , wherein the antibody is conjugated with magnetic beads. 
     
     
         48 . The method of  claim 46 , wherein the protein is a surface protein. 
     
     
         49 . The method of  claim 48 , wherein the surface protein is specific for a particular organ- or tissue-derived microvesicle. 
     
     
         50 . The method of  claim 48 , wherein the surface protein is a major histocompatibility complex protein. 
     
     
         51 . The method of  claim 50 , wherein the major histocompatibility complex protein is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR or a combination thereof. 
     
     
         52 . The method of  claim 48 , wherein the surface protein is FXYD2. 
     
     
         53 . The method of  claim 50 , wherein the biological sample is further enriched comprising contacting the biological sample with magnetic beads conjugated with an antibody specific for the protein FXYD2. 
     
     
         54 . The method of  claim 46  or  117 , wherein the organ- or tissue-derived microvesicles are selected from the group consisting of pancreatic islet-derived microvesicles, kidney-derived microvesicles, islet beta cell-derived microvesicles, kidney-derived microvesicles, donor organ-derived microvesicles, tissue-derived microvesicles, subject organ-derived microvesicles, and tissue-derived microvesicles. 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . A kit, comprising reagents useful for detecting one or more biomarkers in a biological sample of a subject. 
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . The kit of  claim 57 , wherein the reagents specifically bind the biomarker. 
     
     
         61 . The kit of  claim 57 , comprising one or more packaged probe and primer sets, arrays/microarrays, biomarker-specific antibodies or beads. 
     
     
         62 . The kit of  claim 57 , comprising a pair of oligonucleotide primers, suitable for polymerase chain reaction or nucleic acid sequencing, for detecting the one or biomarkers to be identified. 
     
     
         63 . The kit of  claim 57 , comprising at least one monoclonal antibody or antigen-binding fragment thereof, or a polyclonal antibody or antigen-binding fragment thereof, for detecting the one or more biomarkers to be identified. 
     
     
         64 . The kit of  claim 57 , wherein the biomarker is a change in the size and/or number of the donor organ- or tissue-derived microvesicles. 
     
     
         65 . (canceled) 
     
     
         66 . The method of  claim 1 , wherein at least a second biomarker is identified;
 wherein a change in the presence and/or level of the second biomarker allows for monitoring of transplant organ or tissue rejection and/or injury of the subject; and   wherein the second biomarker is selected from the group consisting of: the size, composition, and/or number of recipient T-cell or B-cell microvesicles.   
     
     
         67 . The method of  claim 2 , wherein at least a second biomarker is identified in each of the first and second or subsequence samples;
 wherein the therapy is efficacious for preventing or treating organ or tissue rejection and/or injury in the subject when there is a change in the second biomarker in the second or subsequent samples, relative to the first sample; and   wherein the second biomarker is selected from the group consisting of: the size, composition, and/or number of recipient T-cell or B-cell microvesicles.